Method to produce inactivated w/o emulsion adjuvated vaccines

ABSTRACT

The present invention is directed to a method to prepare an inactivated w/o emulsion adjuvated vaccine, wherein an aqueous solution comprising one or more inactivated angigens is mixed under mild conditions with a ready-made w/o emulsion. Preferably the vaccine is prepared just prior to vaccination. The aqueous solution and w/o emulsion are stirred or shaken by mechanical means or by hand. The present invention also relates to a kit of parts for use in a method according to the invention.

The present invention is related to inactivated vaccines and a method toproduce said vaccines.

Vaccines have been widely used for the prophylaxis and treatment ofinfectious diseases in both animals and man. The vaccines used can bedivided in two main categories: live vaccines and inactivated vaccines.Live vaccines make use of naturally occurring mild strains or attenuatedstrains of live pathogens. Inactivated vaccines comprise antigensconstituting whole inactivated micro-organisms or specific components(subunits) of said micro-organisms. In case of the latter, two types ofinactivated vaccine are distinguished: subunit vaccines in case ofcomponents that have been obtained via biochemical purification, orrecombinant vaccines in case the isolated components of themicroorganism have been prepared via recombinant technology.

Inactivated vaccines have the advantage over live vaccines in that thisway of immunisation against pathogens has no risk of infection. Thegeneral problem perceived with the use of inactivated vaccines is theirinability to raise an immune response that is sufficient for protection.As a consequence, inactivated vaccines are often combined with anadjuvant, i.e. a compound or composition that is capable to increase thegeneral or specific immune response in the vaccinated subject. Theadministration of inactivated micro-organisms or components thereof andthe adjuvant leads to a strong, effective and generally protectiveimmune response. One of the most commonly used adjuvants is awater-in-oil (w/o) emulsion. W/o emulsions provide a two phase systemfor the vaccine: an aqueous phase in which the antigen can be dissolvedor suspended and an oil phase in which the aqueous phase is dispersed assmall droplets.

Inactivated vaccines that make use of a w/o emulsion as adjuvant areusually prepared by emulsifying an aqueous solution comprising theinactivated antigen, a suitable oil and emulsifying agents until a w/oemulsion is obtained in which the antigens are homogeneously distributedover the aqueous phase. The production of these inactivated w/o emulsionadjuvated vaccines takes considerable time and costs, but is generallyregarded as necessary: only thorough emulsification of the antigen wouldlead to a homogeneous distribution of said antigen in the w/o emulsion.This is generally considered to be necessary for effectively stimulatingthe immune response. The emulsification process however is a highlyenergetic process carried out under vigorous process conditionsincluding high temperatures and/or strong shear forces. For someantigens, these vigorous emulsification conditions can alter thestructure or conformation of the antigen and as such reduce the efficacyof the resulting vaccine. Storage in emulsified form can decrease thestability of the antigen because the antigen is present in a dissolvedor suspended state. Furthermore, during storage the chemical componentsthat are. present in the emulsion can reduce the stability of theantigen, as is the case for example with enveloped viruses: prolongedcontact with the emulsifying agent destroys the viral envelope resultingin a decreased efficacy of the vaccine.

Surprisingly it was now found that inactivated, w/o emulsion adjuvatedvaccines can be prepared by simply mixing of an aqueous solutioncomprising the inactivate,d antigen and a ready-made w/o emulsion, saidmixing carried out under mild conditions. The vaccines thus obtainedwere found to be homogeneous compositions, which remained homogeneousfor extended periods of time, despite the fact that the aqueous solutioncomprising the inactivated antigen had not been subjected as a whole toan emulsification process.

As already indicated emulsification is a highly energetic process inwhich an aqueous phase, an, emulsifying (surfactant) agent and anon-aqueous phase, preferably an oil phase, are mixed under vigorousprocess conditions. The mixing process according to the presentinvention however is carried out under extremely mild process conditionscompared to the process conditions of the emulsification process, saidmild conditions including low or no shear forces and ambienttemperatures. Preferably the mixing process according to the presentinvention, is carried out by hand at ambient temperature.

Mixing of said aqueous antigen solution and said ready-made w/o emulsionaccording to the invention can be carried out by mere shaking, stirringor any other way of combining the two constituents without the excessiveprocess parameters that are necessary for emulsification. Compared withthe time needed for emulsification, mixing according to the inventioncan be carried out in a short time period. Preferably the mixing isachieved in a few seconds or minutes. More surprisingly, it was foundthat the mere shaking by hand of said aqueous solution and the alreadyprepared w/o emulsion was sufficient to obtain a stable and homogeneousinactivated vaccine composition with an efficacy similar to aninactivated vaccine prepared according to a standard emulsificationprocess.

The aqueous solution according to the invention comprises one or moreinactivated antigens. An aqueous solution comprising two or moredifferent inactivated antigens can be used in the method according tothe invention to obtain multivalent inactivated vaccine emulsions.

The method of preparation according to the invention has severaladvantages over standard methods of preparation: it is easy to carryout, less time consuming and a more economical process. An importantadvantage of the method according to the present invention is the factthat the inactivated antigen is not exposed to the damaging shear forcesand/or high temperatures that occur during the emulsification process:the chemical structure of the antigen is better preserved.

In a specific embodiment of method according to the invention the w/oemulsion adjuvated vaccine is prepared in the field prior tovaccination. The method provides a veterinarian a more flexible andadequate reaction upon field circumstances: in case of an outbreak thenecessary inactivated antigens can be dissolved in an aqueous solution,mixed by hand with a suitable amount of a stable, ready-made w/oemulsion, and subsequently administered to the subject animals. Thus themethod according to the invention provides for a quick and effectivepreparation of inactivated vaccine emulsions in the field just prior touse.

A further advantage of the method according to the invention is that inthis way inactivated antigens which are normally not compatible, can bemixed together prior to vaccination to obtain a multivalent inactivatedvaccine emulsion. Since the incompatible inactivated antigens arecombined just prior to vaccination, they are only present in theemulsion for a short-lived period during which they cannot exert theiradverse effect on each other.

Another advantage of the method according to the invention is that theinactivated antigens can be stored separately in dried form, preferablyin lyophilised form, to prevent deterioration of the components duringstorage. The stability of the inactivated antigens declines much fasterduring storage in solution or suspension than in dried form. Just priorto vaccination the dried components are re-constituted with water intoan aqueous solution. The dried vaccine components furthermore need lessstorage capacity than their corresponding solutions or suspensions.

Thus, the present invention provides for a method to prepare aninactivated, w/o emulsion adjuvated vaccine, wherein an aqueous solutioncomprising one or more inactivated antigens is mixed under mildconditions with a ready-made w/o emulsion. Preferably the aqueoussolution and the w/o emulsion are stirred or shaken. The aqueoussolution and the w/o emulsion can be mixed, preferably stirred orshaken, by mechanical means or by hand, more preferably by hand. Theready-made w/o emulsion may additionally comprise one or more antigens.

In a further embodiment the invention provides for a method to preparean inactivated, w/o emulsion adjuvated vaccine, wherein an aqueoussolution comprising one or more inactivated antigens is mixed with analready prepared w/o emulsion just prior to vaccination. Preferably theaqueous solution and the w/o emulsion are stirred or shaken, morepreferably shaken. The aqueous solution preferably comprises acombination of inactivated antigens. The ready-made w/o emulsion mayadditionally comprise one or more antigens.

In another embodiment the invention provides for a kit of parts that canbe used for the preparation of an inactivated, w/o emulsion adjuvatedvaccine, said kit comprising at least one dosage of inactivated antigenand a separate dosage of a stable w/o emulsion. Preferably said kitcomprises two or more dosages of inactivated antigen, each dosageconstituting a different inactivated antigen. The inactivated antigen ina kit according to the invention is presented in the form of an aqueoussolution or suspension or in a stable freeze-dried form. Preferably theantigen is presented in a stable, freeze dried form such as e.g.lyosphere, powder or tablet, which has to be dissolved in a small volumeof water before use in a method according to the invention. In additionthe separate dosage of w/o emulsion may comprise one or more antigens.

A kit according to the invention is suitable for use in the field andprovides for tailor-made vaccines. Depending on the field circumstances,a proper selection of inactivated vaccines is dissolved in a smallvolume of water, stirred or shaken with a suitable amount of w/oemulsion and subsequently administered to the subjects. Thus a kitaccording to the present invention enables an adequate and quickreaction to an outbreak in the field.

A preferred kit according to the invention comprises inactivated IBDVantigens and optionally one or more inactivated antigens of chickenpathogens, preferably NDV and IBV.

For the purpose of the invention, the inactivated antigens are definedas immunogenic material derived from infectious micro-organisms(pathogens), which immunogenic material is non-replicative as opposed tolive antigens. Inactivated antigens that are suitable for use in themethod or kit according to the invention include inactivated wholemicro-organisms of viral, bacterial or parasital origin, extracts ofsaid micro-organisms or purified subunits of said micro-organisms.Examples of suitable micro-organisms include but are not limited toNewcastle Disease Virus (NDV), Infectious Bursal Disease Virus (IBDV),Infectious Bronchitis virus (IBV), E. coli, CM, Reo virus, coccidialagents and the like. The micro-organisms can be inactivated by chemicalor physical means following conventional methods. Suitable chemicalagents are for example formaldehyde, glutaraldehyde, β-propiolactone,ethyleneimine and derivatives. Suitable physical agents for inactivationof micro-organisms are for example UV radiation, γ-radiation,“heat-shock”, and X-radiation. The inactivated antigens can also beisolated (recombinant) components or subunits or extracts of a pathogen,e.g. purified protein, protein-polysaccharide,protein-lipopolysaccharides, lipopolysaccharides and the like.

The inactivated antigens according to the invention may be produced byconventional methods known in the art including biochemical purificationor recombinant DNA technology or may be purchased from commercialsources. In case of the latter, the inactivatede antigens are oftenpresent as a freeze-fried or lyophilised powder, tablet or lyosphere,and have to be dissolved in a small volume of water before use in amethod according to the invention.

W/o emulsions that are suitable for use in a method according to theinvention can be produced by methods known in the art or may bepurchased commercially.

The oil component of the w/o emulsion will be present in an amount from40% to 90% by weight, preferably 50% to 80% by weight. Especiallypreferred are w/o emulsions in which the oil component is present in anamount of 55% by weight.

The oil component of the w/o emulsion includes mineral oils such asBayol® and Drakeol® and metabolizable oils. Suitable metabolizable oilsare vegetable oils, fish oils, animal oils, tocopherol and tocopherolderivatives, and synthetically produced oils which can be metabolisedand which are not toxic to the subject's body. Sources for vegetableoils include nuts, seeds, and grains. The most commonly available nutoil are peanut oil, soybean oil, coconut oil and olive oil. Seed oilsinclude safflower oil, cottonseed oil, sunflower oil, sesame seed oil,and the like. Grain oil includes oil from cereal grains such as corn,wheat, oats, rye, rice, and the like. Fish oils include oil which can bereadily recover led from fish, such as cod liver oil, shark liver oil,and the like. Suitable animal oils include whale liver oil. Syntheticoils include a number of branched chain oils that are synthesisedbiochemically in 5-carbon isoprene units and which are referred to asterpenoids. Squalene is a branched unsaturated terpenoid that is presentin shark liver oil. Both squalene and its saturated analogue squalaneare preferred oils and like the other fish oils are readily availablefrom commercial sources or may be produced by methods known in the art.

The aqueous component of the w/o emulsion includes water, a buffer,saline, and the like.

W/o emulsions may be. prepared by conventional methods using emulsifyingagents and/or emulsifying surfactant agents including Span 80, Span 85,Arlacel 80, Tween 80 and the like.

The particle size of the droplets of the dispersed phase is less than 20μm, preferably less than 1 μm, more preferably less than 0.5 μm indiameter. Preferably the w/o emulsion according to the invention is astable emulsion.

Optionally, the w/o emulsions according to the invention comprise one ormore antigens.

The inactivated, w/o emulsion adjuvated vaccines thus prepared by themethod according to the invention can be used to vaccinate animals andman against disease causing pathogens. The vaccines can be administeredper oral or via parenteral routes such as for example subcutaneous (sc.)injection, intramusculair (im) injection, intraperitoneal (ip)injection. Other administration routes that are favoured are applicationvia the respiratory tract, e.g. eye drops, nasal drops, aerosol spray.

The following examples are for illustration only and are not to beinterpreted as a limitation of the scope of the invention.

LEGENDS TO THE FIGURES

FIG. 1: Effect of addition of inactivated IBV antigen to a w/o emulsionbased on mineral oil in chickens (example 4).

EXAMPLES Example 1 Addition of Inactivated Newcastle Disease (NDV)Antigen

Preparation of the vaccines: NDV antigen was grown on chicken eggs,harvested and inactivated (NDV antigen was concentrated 32× byultracentrifugation) and used proportionally for the preparation of thevaccines: either 1 ml of concentrated inactivated NDV was added tobefore emulsification of the mineral oil (vaccines obtained this way arereferred to as the “antigen-inside” formulation representing the priorart vaccines) or 1 ml of concentrated inactivated NDV was added to 500ml of a ready-made mineral oil w/o emulsion and shaken by hand for oneminute prior to administration (vaccine obtained in this way arereferred to as “antigen-outside” formulations representing theinvention). The ready-made mineral oil emulsion in some cases comprisedan additional IBV antigen. Groups of 9 four week old female-SPF chickenswere injected intramuscularly with 0.5 ml of the indicated vaccineformulation. Blood samples were taken at times indicated and antibodytiters in serum were determined by means of the haemagglutinationinhibition test (HI). As presented in table 1, the NDV “antigen-outside”vaccine formulation displayed similar antibody profiles as the“antigen-inside” vaccine formulation. The anti-IBV-antibody responses ofthe samples are presented in table 2, and no large differences wereobserved indicating that the addition of NDV antigen had no influence onthe antibody response against the IBV antigen.

TABLE 1 Formu- Anti-NDV-antibody response* Vaccines lation 3 wkpv 6 wkpv9 wkpv 12 wkpv IBV inside 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 NDVinside 7.9 ± 0.9 8.7 ± 0.9 8.3 ± 1.0 9.2 ± 1.0 IBV + NDV inside 7.9 ±0.8 8.6 ± 0.7 7.9 ± 1.0 8.8 ± 0.7 NDV outside 8.3 ± 0.5 9.1 ± 0.6 7.6 ±0.5 8.3 ± 0.7 IBV + NDV outside 8.1 ± 0.3 8.1 ± 0.8 7.6 ± 1.0 8.0 ± 1.1Control — 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 1.3 ± 2.1 *mean ²log HI titer ±standard deviation Antibody response of chickens against inactivated NDVafter vaccination. Vaccines comprise one or more antigens in mineral oilw/o emulsion (example 1); “inside” = IBV or (IBV + inactivated NDV)antigen added before emulsification of mineral oil; “outside” =inactivated NDV antigen added to ready-made mineral oil w/o emulsionwith IBV antigen or ready-made mineral #oil w/o emulsion without IBVantigen; wkpv = weeks post vaccination; control = saline.

TABLE 2 Formu- Anti-IBV-antibody response* Vaccines lation 3 wkpv 6 wkpv9 wkpv 12 wkpv IBV inside 4.9 ± 1.2 6.9 ± 2.0 7.0 ± 1.1 7.2 ± 1.3 NDVinside 4.0 ± 0.0 4.0 ± 0.0 3.9 ± 0.3 3.9 ± 0.3 IBV + NDV inside 5.6 ±1.2 7.4 ± 0.9 6.7 ± 0.9 7.8 ± 1.1 NDV outside 4.0 ± 0.0 3.9 ± 0.3 4.0 ±0.0 3.8 ± 0.5 IBV/NDV outside 5.2 ± 1.7 6.4 ± 1.4 7.0 ± 1.3 7.0 ± 1.4Control — 4.0 ± 0.0 3.9 ± 1.2 3.8 ± 0.4 3.6 ± 0.7 *mean ²log HI titer ±standard deviation Antibody response of chickens against inactivated IBVafter vaccination. Vaccines comprise one or more antigens in mineral oilw/o emulsion (example 1); “inside” = IBV or (IBV + inactivated NDV)antigen added before emulsification of mineral oil; “outside” =inactivated NDV antigen added to a ready-made mineral oil w/o emulsionwith IBV antigens or a ready-made mineral oil #w/o emulsion without IBVantigen; wkpv = weeks post vaccination; control = saline

Example 2 Addition of F11 Antigen

Preparation of the vaccines: F11 pilus protein derived from E. coli wasused to prepare the vaccines similar to the procedure described above:either 1 ml of concentrated F11 antigen was added before emulsificationof the mineral oil (“antigen-inside” formulation) or 1 ml ofconcentrated F11 antigen was added to 500 ml of emulsion and shaken byhand for one minute prior to administration (“antigen-outside”formulation). The ready-made mineral oil emulsion in some casescomprised additional IBV and NDV antigens. Groups of 10 four week oldfemale SPF chickens were injected intramuscularly with the indicatedvaccine formulation (10 μg F11 per dosis vaccine). Blood samples weretaken at times indicated and antibody titers were determined in serum bymeans of an Elisa (incubation with F11 coated microtiter plates andchicken serum; incubation with anti-chicken Ig antibodies-enzymeconjugate). When F11 antigen was added to w/o emulsions without antigenor w/o emulsions comprising IBV+NDV antigens, no significant differencesin antibody response against F11 were observed between the variousgroups (table 3). Moreover, addition of F11 to the emulsions comprisingIBV and NDV antigens had no influence on the antibody response againsteither IBV or NDV (data not shown).

TABLE 3 Formu- Anti-F11-antibody response* Vaccines lation 3 wkpv 6 wkpv9 wkpv 12 wkpv IBV + NDV inside  5.3 ± 0.7  6.0 ± 0.7  7.0 ± 0.8  7.4 ±0.6 F11 inside 10.7 ± 1.8 13.3 ± 2.8 12.6 ± 2.6 14.6 ± 1.9 IBV/NDV/inside  8.7 ± 1.3 11.1 ± 1.6 11.7 ± 1.3 13.0 ± 2.1 F11 F11 outside  9.3± 1.4 12.2 ± 1.1 13.1 ± 1.7 14.9 ± 1.4 IBV/NDV/ outside  8.7 ± 2.2 11.8± 2.2 13.2 ± 1.8 13.2 ± 1.9 F11 control —  5.4 ± 0.7  6.3 ± 0.6  7.2 ±0.8  7.5 ± 0.8 *mean ²log Elisa titer ± standard deviation Antibodyresponse of chickens against F11 pilus protein after vaccination.Vaccines comprise one or more antigens in mineral oil w/o emulsion(example 2); “inside” = F11 and/or (IBV and NDV) antigen added beforeemulsification of mineral oil; “outside” = F11 antigen added toready-made mineral oil w/o emulsion with IBV and NDV antigens orready-made mineral oil w/o emulsion without IBV and NDV antigens; #wkpv= weeks post vaccination; control = saline

Example 3 Addition of Inactivated Gumboro Disease Virus (IBDV)

Preparation of the vaccines: Various vaccines were prepared usingdifferent lots of inactivated IBDV antigens (10^(7.3) TCID₅₀/ml) similarto the procedure described above: either 1 ml of concentratedinactivated IBDV antigen was added before emulsification of the mineraloil (“antigen-inside” formulation) or 1 ml of concentrated inactivatedIBDV antigen was added to 500 ml of ready-made mineral oil w/o emulsionand shaken by hand for one minute prior to administration(“antigen-outside” formulation). Groups of 8 four week old female SPFchickens were injected intramuscularly with the indicated vaccineformulation. Blood samples were taken at times indicated and antibodytiters were determined in serum by means of an Elisa (incubation withIBDV-coated microtiter plates and chicken serum; incubation withanti-chicken-Ig antibodies-enzyme conjugate). When concentratedinactivated IBDV was added to the emulsions (table 4), a significanthigher antibody response at 3 and 6 weeks post vaccination (rapidresponse) was found in groups that received emulsions With IBDV“outside”-formulation compared to the antibody response in the groupsthat received corresponding emulsions with IBDV “inside” formulation. Atlater stages after vaccination no large differences were observed,indicating that in case of vaccination with IBDV “antigen-outside”formulations the antibody response was very rapid compared tovaccination with other “antigens-outside” formulations (see results ofexamples 1 and 2). It can also be concluded that the observed antibodyresponse is not dependent on a particular antigen lot.

Example 4 Addition of Inactivated Infectious Bronchitis Virus (IBV)

Preparation of the vaccines: Vaccines were prepared with inactivated IBVantigens similar to the procedure described above: 10 or 100 mlnon-concentrated inactivated IBV antigen were either added beforeemulsification of the mineral oil (“antigen-inside” formulation) oradded to respectively 490 or 400 ml of ready-made mineral oil w/oemulsion and shaken by hand for 1 min prior to administration(“antigen-outside” formulation). Groups of 9 four week old female SPFchickens were injected intramuscularly with the indicated vaccineformulation. The addition of different volumes of inactivated IBVantigen to a mineral oil w/o emulsion resulted in significantly higherantibody responses at 3 weeks post vaccination (pv) compared with theantibody responses observed after vaccination with the “antigen-inside”formulations. At 6 and 12 weeks post vaccination no significantdifferences in response between the three groups were observed (FIG. 1).

TABLE 4 Formu- Anti-F11-antibody response* Vaccines lation 3 wkpv 6 wkpv9 wkpv 12 wkpv IBDV inside  5.4 ± 0.5 10.5 ± 1.8 12.0 ± 1.9 12.4 ± 1.7IBDV inside  5.3 ± 0.7  8.9 ± 2.0 10.1 ± 1.6 10.6 ± 1.0 IBDV inside  5.7± 0.8 10.6 ± 2.3 11.3 ± 2.6 11.4 ± 2.1 IBDV inside  6.0 ± 1.0  9.5 ± 2.011.3 ± 1.2 12.0 ± 1.0 IBDV inside  6.5 ± 1.7 11.4 ± 1.1 12.3 ± 1.3 11.7± 1.6 IBDV inside  7.2 ± 2.3 10.7 ± 2.7 12.0 ± 2.3 12.1 ± 2.0 IBDVinside  5.8 ± 0.8 10.8 ± 1.6 11.5 ± 1.6 11.2 ± 1.8 IBDV inside  5.4 ±1.1  8.3 ± 2.0 10.3 ± 1.6 10.9 ± 1.6 IBDV outside  8.3 ± 1.8 12.1 ± 1.112.8 ± 1.3 11.9 ± 1.4 IBDV outside  7.9 ± 2.5 11.6 ± 1.3 12.0 ± 1.2 11.7± 1.3 IBDV outside  7.8 ± 1.2 12.1 ± 1.3 12.4 ± 1.6 11.4 ± 2.1 IBDVoutside 10.4 ± 1.5 11.7 ± 1.0 11.6 ± 1.4 10.9 ± 1.6 IBDV outside  9.1 ±0.9 12.8 ± 1.1 13.3 ± 0.8 12.2 ± 1.0 IBDV outside  8.4 ± 1.6 11.6 ± 1.312.7 ± 0.9 12.3 ± 0.6 IBDV outside  7.6 ± 1.4 12.0 ± 1.7 12.7 ± 2.2 12.1± 1.5 IBDV outside  7.5 ± 1.8 10.7 ± 1.3 11.3 ± 1.9 10.8 ± 2.1 Control — 5.3 ± 0.5  5.0 ± 0.0  5.1 ± 0.3  5.1 ± 0.3 *mean ²log Elisa titer ±standard deviation Antibody response of chickens against IBDV aftervaccination. Vaccines comprising the antigens in mineral oil w/oemulsion; “inside” = inactivated IBDV antigen added beforeemulsification of mineral oil; “outside” = inactivated IBDV antigenadded to ready-made mineral w/o emulsion without antigens; wkpv = weekspost vaccination; control = saline.

Example 5 Storage of w/o Emulsions to Which Inactivated NDV was Added

Vaccines prepared according to example 1 were stored for 3 months at +4°C. before vaccination. The same procedure as described in Example 1 wascarried out and the results are shown in table 5. No significantdifferences in antibody response were noted between vaccination with“antigen-outside” formulations and vaccination with “antigen-inside”formulations, indicating that even after storage w/o emulsions to whichinactivated antigen was added after emulsification performed similar tothose w/o emulsions to which the inactivated antigen was added beforeemulsification and that the homogeneity of the emulsion was not altered.

TABLE 5 Formu- Anti-F11-antibody response* Vaccines lation 3 wkpv 6 wkpv9 wkpv 12 wkpv NDV inside 7.4 ± 0.5 7.6 ± 0.5 7.6 ± 0.7 7.0 ± 0.9 NDVoutside 8.3 ± 0.5 8.6 ± 0.9 7.8 ± 0.8 7.1 ± 0.8 IBV + NDV inside 8.0 ±1.0 9.0 ± 1.2 8.4 ± 0.9 8.2 ± 1.0 IBV + NDV outside 8.1 ± 0.8 8.7 ± 1.08.4 ± 1.0 7.4 ± 0.7 Control — 0.0 ± 0.0 0.1 ± 0.3 0.0 ± 0.0 0.1 ± 0.3*mean ²log HI titer ± standard deviation Antibody response of chickensagainst inactivated NDV after vaccination with vaccines. Vaccinescomprise one or more antigens in mineral oil w/o emulsion and have beenstored for 3 months at 4° C. before use in vaccination; “inside” = IBVor (IBV + inactivated NDV) antigen added before emulsification ofmineral oil; “outside” = inactivated NDV antigen added to ready-mademineral #oil w/o emulsion with IBV antigen or ready-made mineral oil w/oemulsion without IBV antigen; wkpv = weeks post vaccination; control =saline.

What is claimed is:
 1. A method for preparing an inactivated,water-in-oil emulsion adjuvated vaccine, comprising mixing an aqueoussolution comprising one or more inactivated antigens under mildconditions with a ready-made water-in-oil emulsion.
 2. The methodaccording to claim 1, wherein said aqueous solution and said ready-madewater-in-oil emulsion are mixed just prior to vaccination.
 3. The methodaccording to claim 1, wherein said antigen comprises inactivatedInfectious Bursal Disease Virus (IBDV) or immunogenic subunits thereof.4. The method according to claim 1, wherein the ready-made water-in-oilemulsion comprises at least one antigen.
 5. A kit of parts that can beused for the preparation of an inactivated water-in-oil emulsionadjuvated vaccine just prior to vaccination, said kit comprising atleast one dosage of inactivated antigen and a separate dosage ofwater-in-oil emulsion.
 6. The kit according to claim 5, comprising twoor more dosages of inactivated antigen, each dosage constituting adifferent inactivated antigen.
 7. The kit according to claim 5, whereinat least one of the inactivated antigens is inactivated IBDV antigen. 8.The kit according to claim 5, wherein the water-in-oil emulsioncomprises at least one antigen.